Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

Izabela Sokolowska,Cristina Dorobantu,Norica Branza-Nichita,Alina Macovei,Alisa G Woods,Costel C Darie

doi:10.1186/1477-5956-10-47

Abstract

Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (−) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (−) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.

Highlights

The hepatitis B virus (HBV) is a noncytopathic, hepatotropic DNA virus of the Hepadnaviridae family [1]
Using Annexin proteins as example, we looked at both the Mascot scores and emPAI scores for these proteins, as well as for the number of peptides identified per protein per condition in the database search, as well as direct comparison of the intensities of the precursor ions that correspond to the same peptide and for which Mass spectrometry (MS)/MS was observed in the same protein in both (−) and (+) conditions
Concluding remarks In this study, we used the HepaRG cells to investigate the differences between the protein content of the plasma membranes from differentiated and undifferentiated cells

Summary

Introduction

The hepatitis B virus (HBV) is a noncytopathic, hepatotropic DNA virus of the Hepadnaviridae family [1]. While our understanding of HBV replication and assembly has advanced considerably in the last years, the early steps of the viral life cycle are still a matter of debate. This is mainly a consequence of the poor in vitro infectivity systems available, which until recently were based on primary human and chimpanzee hepatocytes [3]. Their accessibility is limited and the level of HBV replication is low, which makes the experimental data often difficult to interpret. Quantification of RNA levels within the whole population of differentiated cells showed high expression of adult hepatocytes-specific markers, such as albumin and aldolase B mRNAs, while the detoxification enzymes cytochrome P450, CYP 2E1 and CYP 3A4 were up-regulated in cells undergoing trabecular organization

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Conclusion